RESEARCH ARTICLE 4353
Development 140, 4353-4361 (2013) doi:10.1242/dev.096925
© 2013. Published by The Company of Biologists Ltd
Switching cell fates in the developing Drosophila eye
Yannis Emmanuel Mavromatakis and Andrew Tomlinson*
SUMMARY
The developing Drosophila ommatidium is characterized by two distinct waves of pattern formation. In the first wave, a precluster
of five cells is formed by a complex cellular interaction mechanism. In the second wave, cells are systematically recruited to the cluster
and directed to their fates by developmental cues presented by differentiating precluster cells. These developmental cues are mediated
through the receptor tyrosine kinase (RTK) and Notch (N) signaling pathways and their combined activities are crucial in specifying
cell type. The transcription factor Lozenge (Lz) is expressed exclusively in second wave cells. Here, we ectopically supply Lz to precluster
cells and concomitantly supply the various RTK/N codes that specify each of three second wave cell fates. We thereby reproduce
molecular markers of each of the second wave cell types in precluster cells and draw three inferences. First, we confirm that Lz
provides key intrinsic information to second wave cells. We can now combine this with the RTK/N signaling to provide a cell fate
specification code that entails both extrinsic and intrinsic information. Second, the reproduction of each second wave cell type in the
precluster confirms the accuracy of the RTK/N signaling code. Third, RTK/N signaling and Lz need only be presented to the cells for a
short period of time in order to specify their fate.
INTRODUCTION
When cells make developmental decisions, two influences can play
crucial roles: the external information that is presented to cells, and
the internal information that determines how the cells respond to it.
The external information can be of diverse molecular nature but is
usually in the form of secreted peptides that diffuse to target cells,
or integral membrane proteins presented by immediate neighbors.
The internal information is usually encoded by transcription factors
that determine how the cells respond to these signals.
The developing Drosophila ommatidium provides an excellent
model system with which to study how extrinsic and intrinsic
information is integrated to deliver clear cellular developmental
directives. Here, two classes of signaling pathway relay the external
information: the receptor tyrosine kinase (RTK) and Notch (N)
signaling pathways. The RTK pathway uses two distinct classes of
signals. Spitz, which acts as the ligand for the Drosophila EGF
receptor (DER; Egfr – FlyBase), is a diffusible peptide (Freeman,
1994; Freeman, 1996; Kumar et al., 1998), whereas Bride of
sevenless (Boss) is an integral membrane protein that activates the
Sevenless (Sev) RTK in immediately adjacent cells (Hafen et al.,
1987; Krämer et al., 1991; Reinke and Zipursky, 1988). The N ligand
in the developing eye is Delta (Dl), another integral membrane
protein, which activates N only in direct neighbors (ArtavanisTsakonas et al., 1995; Parks et al., 1995). The intrinsic information
comes from the developmental history of the retinal tissue, in which
a complex transcription factor web (Kumar, 2010) defines the tissue
as ‘eye’, and when the cells receive a fate directive they interpret that
signal as an instruction to make one of the many ommatidial cells.
The ommatidium is constructed in two distinct waves. First, a
group of cells exits the cell cycle, undergoes a complex interaction
and generates the precluster comprising prospective photoreceptors
Department of Genetics and Development, College of Physicians and Surgeons,
Columbia University, 701 West 168th Street, New York, NY 10032, USA.
*Author for correspondence ([email protected])
Accepted 9 August 2013
R2,3,4,5,8. In the second wave, cells are systematically incorporated
into the cluster and the unit grows by simple accretion (Ready et
al., 1976; Tomlinson and Ready, 1987). This is a reiterative process,
in which cells are first recruited into specific positions within the
cluster and then developmental signals from the differentiating
cluster cells direct the fate of these new additions. As these cells
differentiate they create new positions for cell recruitment and the
process is repeated (Fig. 1A). We focus our studies on the fate
specification of the first seven cells that are incorporated during the
second wave in three rounds of recruitment. The first three cells that
join the cluster are directed to become photoreceptors – two of the
R1/6 generic class and one of the specialized R7 type. Next, two
rounds of recruitment incorporate two pairs of cells, all four of
which are directed to become lens-secreting cone cells. Thus, in this
process three cell types are specified: the R1/6 photoreceptors, the
R7 photoreceptor, and the cone cells (Fig. 1B).
A series of experiments has suggested that two binary molecular
switches lie at the heart of the cell fate specifications of the second
wave cells (Fig. 1C). RTK signaling determines whether Tramtrack
88 (Ttk), a transcription factor that represses photoreceptor
development, is degraded (Li et al., 1997; Li et al., 2002; Tang et al.,
1997). If Ttk is degraded then a second wave cell becomes a
photoreceptor. If not, the photoreceptor fate is repressed and a cone
cell is specified. The second binary switch relates to N activity, such
that if Ttk is degraded (the photoreceptor fate) and the cell has low
N activity it becomes an R1/6 photoreceptor, and if N activity is
high it becomes an R7 photoreceptor (Fig. 1C) (Tomlinson et al.,
2011).
Work from the Banerjee laboratory determined in a series of
elegant experiments that the transcription factor Lozenge (Lz), is
expressed exclusively in second wave cells (expression is absent
from the precluster) (Flores et al., 1998) and that ectopic expression
of Lz in precluster cells generates ectopic R7s (Daga et al., 1996),
suggesting that supplying Lz to precluster cells redirects their fate
responses to those of the second wave cells. Thus, Lz appears to act
as the intrinsic factor that determines whether RTK and N signals
are interpreted as precluster or second wave fate signals. In this
paper, we expand upon this insight and demonstrate that the three
Development
KEY WORDS: Drosophila, R7, Cell fate, Eye, Lozenge
characterized second wave fates can be systematically reproduced
in the R3/4 precursors of the precluster when Lz is concomitantly
supplied. Specifically, we show that when Ttk is absent and N
activity is high, R7s are specified; when Ttk is absent and N activity
is low the R1/6 class is formed; and when Ttk is present the cone
cell fate is specified. This allows us to now incorporate Lz into the
RTK/N/Ttk code of second wave fates, and thereby combine the
intrinsic with the extrinsic information.
Additionally, this work provides cogent support for the signaling
code of the second wave cells. Originally, the roles of RTK, N and
Ttk in the specification of the second wave types were inferred from
experimental manipulations of the second wave cells, and although
all results were internally consistent, the complexity of the RTK and
N interactions was somewhat surprising, and we sought another
method for independent verification of this code. The use of
precluster cells to reiterate second wave fates has now provided that
verification; each of the cell types appears to be specified in the Lzexpressing R3/4 precursors in exactly the manner predicted by the
cell fate codes. Thus, as we add the intrinsic information, we add it
to a newly validated extrinsic signaling code.
It has been clear for some time that the signals that specify the
second wave cells are presented to the cells during a short time
window. In this work, we establish that the Lz information is similarly
only required for a short time period, and we infer that both the
intrinsic and extrinsic information need only be presented briefly to
the cells for them to lock-in their final cell fates. This raises the
intriguing question of the molecular mechanism by which the
ephemeral specification information is converted into stable cell fate
directives that guide the differentiation and maturation of the cells.
MATERIALS AND METHODS
Immunohistochemistry and histology
Protocols for adult eye sectioning and antibody staining have been described
previously (Tomlinson et al., 2011). Primary antibodies: rabbit anti-β-gal
(Cappel); rabbit anti-GFP, mouse anti-GFP IgG2a (Molecular Probes);
guinea pig anti-Runt (gift of J. Reinitz, Stony Brook University, NY, USA);
rabbit anti-Runt (gift of A. Brand, Gurdon Institute, Cambridge, UK);
guinea pig anti-Sens (gift of H. Bellen, Baylor College of Medicine,
Houston TX, USA); rabbit anti-Bar (gift K. Saigo, Kobe University, Japan);
rat anti-Bar (gift of T. Cook, University of Cincinnati School of Medicine,
USA); mouse anti-Svp (gift of Y. Hiromi, National Institute of Genetics,
Japan); rabbit anti-RFP (MBL International Corporation); rat anti-Elav and
mouse anti-Cut (Developmental Studies Hybridoma Bank). Alexa Fluor
488, 555 and 647 conjugated secondary antibodies were used (Molecular
Probes). For analysis of adult eye sections, at least 200 individual ommatidia
were scored for each genotype. A minimum of 40 individual eye imaginal
discs were analyzed for each genotype.
Identification of individual cells in third larval instar eye discs
In wild type ommatidia cells join the ommatidium in a stereotypical
manner, they adopt specific positions in the cluster and they express
molecular markers characteristic of their fate. We use a panel of antibodies
to detect these fates, including Runt, Sens, Elav, Ro, Bar and Cut. All
photoreceptors express Elav; in addition, R8 expresses Runt and Sens,
R3/4s express Svp, R1/6s express Bar and Svp (at lower levels than R3/4)
and R7s express Runt. Cone cells express Cut (without Elav). When
examining mutant ommatidia we track cells as they join the ommatidia
and occupy specific positions, and then determine which molecular
markers they subsequently express.
Fly stocks
The following lines were used: sev[d2], sev.N[intra] (Fortini et al., 1993);
sev.RapV12 (Mavromatakis and Tomlinson, 2012); lz77a7 (Batterham et al.,
1996); sev.Su(H)enR (Tomlinson and Struhl, 2001); Rh3;Rh4 lacZ (Pan-R7
lacZ) (Hofmeyer et al., 2006).
Development 140 (21)
Generation and transformation of constructs
The coding sequences for DsRed, phyl and lz were each amplified using
appropriate PCR primers and cloned into an attB vector containing 2× sev
enhancer elements and the sev promoter element. For ro.GFP and ro.Gal4
a 1.9 kb NotI-EcoRV (5204-7132) fragment of rough genomic DNA was
cloned upstream of the minimal hsp70 promoter and then cloned into an
attB vector containing either GFP or Gal4 coding sequence. Transformation
of all the above attB plasmids was carried out following standard protocols
(Bischof et al., 2007).
RESULTS
The ectopic expression of Lz in R3/4 precluster
cells specifies them as R7s
The sev enhancer element (Basler et al., 1991) drives expression at
high levels in R3/4, R7 and cone cell precursors (Fig. 1D), and to
target Lz expression to the R3/4 precursor we engineered and
transformed a sev.lz construct. We observed [as previously described
(Daga et al., 1996)] that the cell in the R4 position frequently
transformed into an R7-type cell, as evidenced by cellular
morphology in adult sections (Fig. 2D, red arrowheads), and by the
expression of specific molecular markers in third larval instar discs
(Fig. 2E). Less frequently, both R3/4 precursors transformed into
R7s (Fig. 2D, red circle), and occasionally the R3 precursor
transformed into the R7 type when the R4 precursor did not (Fig.
2D, green arrowhead).
The R3/4 cells of the normal precluster undergo an N-Dl
interaction that results in one cell with high N activity (R4) and the
other with relatively low N activity (R3) (Cooper and Bray, 1999;
Fanto and Mlodzik, 1999; Tomlinson and Struhl, 1999). Since the
cell in the R4 position experiences high N activity, then the frequent
specification of this cell as an R7 was consistent with the cell fate
code for R7; it requires high N levels whereas the R1/6 types are
specified by low N activity (Fig. 1C). R3/4 types are
morphologically indistinguishable from R1/6 cells in adult sections
but have a clear molecular signature in terms of the transcription
factors that they display during the development of the eye disc:
R3/4 express Svp whereas R1/6 cells express both Svp and Bar. The
cells in the R3 positions in sev.lz eye discs did not express Bar,
suggesting that although the high levels of N activity specified R7s,
the lower level of N activity did not specify R1/6 types. We return
to the specification of the R1/6 type below.
Raising N activity in the R3/4 precursors specifies
both as R7s in the presence of Lz
To test whether it was indeed the high levels of N activity in R4
precursors that specified them as R7s, we concomitantly expressed
activated N (N*) in both the R3 and R4 cells using a sev.N*
construct (Fortini et al., 1993; Tomlinson and Struhl, 2001) (sev.N*;
sev.lz). sev.N* has a number of effects on second wave cells, two of
which are pertinent here. First, it directs the R1/6 cells to the R7
fate, thereby generating three R7 cells in the R1/6/7 positions (Fig.
2G-I) (Cooper and Bray, 2000; Tomlinson and Struhl, 2001).
Second, it can cause one or more of these three cells to be lost
shortly thereafter, resulting in adult ommatidia with one, two or
three R7 cells in this position (Tomlinson and Struhl, 2001). It
should be noted that supplying N* to the R3/4 precursor using
sev.N* has no effect on their general cell fate specification (Fig. 2H).
When we examined sev.N*; sev.lz adult eye sections the ommatidia
were randomly rotated, preventing the unambiguous identification
of the R1/6/7 and R3/4 sides of the ommatidia. Although there was
variability in structure, ommatidia were frequently observed
containing two large rhabdomere cells and four small R7-like cells
Development
4354 RESEARCH ARTICLE
Switching cell fates
RESEARCH ARTICLE 4355
(Fig. 2J, red arrowheads). Thus, in sev.N*; sev.lz eyes, in addition to
the R1/6/7 cells, the R3/4 cells now appeared as R7s. This was
corroborated by examination of larval eye discs in which we
observed cells in the R3/4 positions now expressing R7 markers
(Fig. 2K). These results suggest that, if an R3/4 precursor cell
expresses Lz and has high N activity, then it is specified as an R7
cell.
In each of these experiments we identified the R7s in adult
sections by their small rhabdomeres and in the eye discs by their
expression of Runt. R8s share both these features, and to ensure that
the cells in the eye disc and adult retina were indeed R7s we
performed two controls. R8 precursors in the eye disc are uniquely
identified by expressing both Runt and Sens, whereas R7 precursors
express only Runt. In all experiments in which ectopic Runtexpressing cells were detected, we counterstained to ensure that they
were not also expressing Sens (an example is shown in Fig. 2E⬘). To
determine whether the supernumerary small rhabdomere cells
detected in adult sections were indeed of the R7 type, we monitored
the transcriptional activity of the opsin genes that are typically
expressed in these cells using the combined Rh3.lacZ and Rh4.lacZ
transgenes (Hofmeyer et al., 2006). In wild-type adult ommatidia all
R7s were positively stained (Fig. 3A, red arrowheads), as were the
supernumerary small rhabdomere cells in sev.lz (Fig. 3B) and sev.lz;
sev.N* (Fig. 3C) eyes. Thus, by examining molecular markers in
the eye disc and by morphology and opsin expression in the adult,
we corroborated that R3/4 precursors could be transformed into
bona fide R7 photoreceptors by the ectopic expression of Lz.
The role of Sev in specifying precluster R7s
Above, we have discussed the role played by N in specifying the
R7 versus R1/6 photoreceptor types once Ttk is degraded (Fig.
1C), but prior to this N plays two roles in determining whether
Ttk is degraded or not. These two roles are, surprisingly,
antagonistic. One function is to antagonize RTK signaling and
thereby prevent DER activity from degrading Ttk. The other is to
activate sev transcription and supply a high level of this RTK to
the R7 precursor, following which, the combined actions of Sev
and DER suffice to overcome the N-induced block to the pathway,
such that Ttk is degraded and the cell is specified as a
photoreceptor. Indeed, in the sev0 mutant, the R7 precursor
becomes a cone cell because, in the absence of Sev, there is
insufficient RTK activity to overcome the inhibition imposed by
N, and this therefore defines a key feature of second wave cells:
if they experience high N activation and are unable to activate Sev,
then Ttk will persist and they will become cone cells. We therefore
tested whether R7s specified in the precluster by ectopic Lz
expression were Sev dependent and whether they became cone
cells in its absence. Key to this experiment is the fact that the R3/4
precursors express high levels of Sev and directly contact the
Boss-expressing R8 cell, so the Sev ligand is therefore directly
available to them just as it is to the native R7 (Fig. 1D).
When Sev was removed from sev.lz eyes (sev0; sev.lz) we inferred
the constitutive loss of endogenous R7 and that any remaining R7s
would arise from the R3/4 precursors. Examination of adult sections
showed a frequent absence of R7-like cells from the ommatidia, and
that the predominant phenotype was ommatidia containing five
large rhabdomere cells (Fig. 4A, inset). However, in a minority of
ommatidia, R7-like cells were clearly present. To investigate this
further we examined sev0; sev.lz eye discs. As expected, the
endogenous R7 cell was specified as a cone cell, labeled with Cut,
as were most of the cells in the R4 position (Fig. 3B). But,
correlating with the adult investigation, in some ommatidia, cells in
the R3/4 positions expressed R7-specific markers. Interestingly,
some of these cells also expressed the cone cell marker Cut (not
Development
Fig. 1. Summary of the sequence of cell incorporation, cell fate specification and marker expression in the developing Drosophila
ommatidium. (A) The incorporation and differentiation of the first seven cells to be added to the precluster. (i) The precluster (R2,3,4,5,8), is surrounded
by a ‘sea’ of undifferentiated second wave cells (gray ovals). (ii) Three cells from the pool join the precluster on the R2/5/8 face. (iii) Two cells begin to
differentiate as R1/6 photoreceptors while the R7 precursor between them delays differentiation and two cone cell precursors (C) join the cluster at the
flanks. (iv) The R7 precursor begins to differentiate and two additional cone cell precursors join the cluster. (v) The differentiation of the cone cells ends
this phase of ommatidial development with all seven of the newly added cells differentiating as specific cell types. (B) The three different cell types
(R1/6, R7 and cone cells) that are added to the precluster. (C) The cell fate code for the R1/6, R7 and cone cells. If a cell degrades Ttk and has high N
activity it becomes an R7, but if it has low N activity it becomes an R1/6 type. If the cell fails to degrade Ttk it becomes a cone cell. (D) The expression
patterns of Sev and its ligand Boss.
4356 RESEARCH ARTICLE
Development 140 (21)
Fig. 2. The effects of expressing Lz in R3/4
precursors. (A) Section through an adult wild-type
eye. Inset illustrates the cell arrangement in a single
ommatidium. (B) A wild-type third instar eye disc
showing expression of Svp (red) in the R3/4 and R1/6
precursors and Runt (blue) in R7. (C) Schematic
representation of cluster cell identities. (D) Section
through an adult sev.lz eye. Cells in the R4 position
frequently transform into R7-like cells (red
arrowheads), less frequently both R3/4 cells appear as
R7s (red circle), and occasionally the cell in the R3
position appears as an R7 (green arrowhead). Inset
shows a typical sev.lz ommatidium with the cell in the
R4 position appearing as an R7. (E) A sev.lz third instar
disc labeled for Svp and Runt. The cells in the R4
position frequently display the R7 marker Runt.
(E⬘) A single ommatidium stained for Sens (green),
Runt and Svp. Runt is expressed in both R7 and R8,
whereas Sens is restricted to R8. The Runt-expressing
cell in the R4 position does not express Sens.
(F) Summary of the most frequent sev.lz fates.
(G) Section through an adult sev.N* eye. Typically, two
or three R7-like cells (arrowheads) are observed in the
R1/6/7 positions caused by the transformation of R1/6
precursors to R7s accompanied by the loss, or not, of
one or more of the R7s. (H) A sev.N* third instar eye
disc showing the cells in the R1/6/7 positions
differentiating as R7s, evidenced by the expression of
Runt and the absence of Svp. (I) Summary of sev.N*
ommatidia indicating the transformation of R1/6
precursors to the R7 fate. (J) Section through an adult
sev.lz; sev.N* eye. Ommatidia typically contain two large
rhabdomere cells and four R7-like cells (arrowheads).
(K) A sev.lz; sev.N* third instar eye disc showing the
potent transformation of R3/4 and R1/6 into R7 cells.
The cell marked 1 is an infrequent R1/6 cell that failed
to transform. (L) Schematic representation of a typical
sev.lz; sev.N* ommatidium.
sev.lz), and found that Sev was now critically required. Here, adult
ommatidia frequently showed only two large rhabdomere cells
(inferred as R2/5), and a small rhabdomere cell that appeared
morphologically to be R8, suggesting that none of the R1/6/7 or
R3/4 precursors differentiated as photoreceptors (Fig. 4D). To
validate this interpretation and to determine the fate of these cells
Fig. 3. Rh3;Rh4-lacZ reporter
activity in wild-type and
experimental adult eyes sections.
(A) A wild-type eye showing lacZ
reporter staining of R7s (blue,
arrowheads). (B) In sev.lz eyes the
extra R7-like cells are all labeled
(arrowheads). (C) In sev.lz; sev.N*
eyes the four R7-like cells are all
labeled (arrowheads).
Development
shown) suggesting that they were in an ambiguous cell state – a
condition not observed in R4 precursors of sev.lz flies. From these
results we infer that there is a partial dependence on Sev for the
specification of the sev.lz R3/4 precursors as R7s.
We next examined the Sev dependence of R3/4-to-R7
transformations when N* was additionally supplied (sev0; sev.N*;
Switching cell fates
RESEARCH ARTICLE 4357
Fig. 4. The role of Sev in sev.lz R3/4 precursors.
(A) Section through a sev0; sev.lz eye. Ommatidia
containing five outer photoreceptors (inset) are often
seen. In ommatidia in which there are two small
rhabdomeres, we infer the presence of supernumerary
R7s. (B) A sev0; sev.lz third instar larval disc stained for
Elav, Runt and Cut. The cells in the R4 position most
frequently express Cut. Occasionally they express Runt.
(C) Schematic representation of the most common sev0;
sev.lz ommatidium. (D) Section through a sev0; sev.lz;
sev.N* eye. Ommatidia containing only two large
rhabdomere cells are frequently observed with an R8like small rhabdomere cell. (E,E⬘) In sev0; sev.lz; sev.N*
third instar eye discs the R8 (Runt/Elav expression) and
R2/5 (Elav expression) precursors differentiate normally,
whereas the R3/4, R1/6 and R7 precursors differentiate
as cone cells (Cut expression). (F) The fates of a sev0;
sev.lz; sev.N* ommatidium. (G) Section through a
sev.phyl eye showing the multiple R7 phenotype. (H) An
apical image at the cone cell level of a sev.phyl third
instar eye disc showing cells in the anterior and
posterior cone cell positions differentiating as R7s along
with the native R7. (I) Schematic representation of a
typical sev.phyl ommatidium. (J) Section through a sev0;
sev-lz; sev.N*; sev.phyl eye, showing four R7-like cells
(arrowheads) and two large rhabdomere cells. (K) A
sev0; sev.lz; sev.N*; sev.phyl third instar eye disc, showing
R3/4 and R1/6 cells frequently specified as R7s. (L) A
sev0; sev.lz; sev.N*; sev.Rap* third instar eye disc showing
the same phenotype as sev0; sev.lz; sev.N*; sev.phyl.
(M) Schematic representation of a sev0; sev.lz; sev.N*;
sev.phyl or sev0; sev.lz; sev.N*; sev.Rap* ommatidium.
High-level RTK activation can substitute for Sev in
R3/4-to-R7 specification
Above we ascertained that when R3/4 precursors express Lz and have
high N activity they critically require Sev for their specification as
R7s; in its absence the cells fail to degrade Ttk and instead
differentiate as cone cells. We inferred that Sev was providing the
high RTK activity required to degrade Ttk and specify the
photoreceptor fate. If this were correct, then supplying an independent
high-level activator of the RTK pathway should rescue the absence of
Sev. We used sev.rapV12, which we previously showed to be a potent
activator of the RTK pathway (Mavromatakis and Tomlinson, 2012),
and examined sev0; sev.N*; sev.lz; sev.rapV12 animals. The adult eyes
of this genotype were too disorganized for any meaningful evaluation,
but in third instar discs the R3/4 precursors now reverted back to the
R7 fate (Fig. 4L). Thus, high-level RTK pathway activation is able to
substitute for the absence of Sev. This suggests that the role of Sev in
Lz-expressing R3/4 precursors counteracts the photoreceptor
inhibiting activity of high level N activity by providing a potent
activation of the RTK pathway.
Transcriptional activation of phyl can substitute
for Sev in R3/4-to-R7 specification
In the native R7 precursor, the high-level RTK activity provided by
Sev overcomes the N-imposed block on photoreceptor fate by
activating the transcription of phyl. Phyl then acts to trigger Ttk
degradation by linking it to the Sina E3 ubiquitin ligase (Li et al.,
Development
we stained sev0; sev.N*; sev.lz third instar discs for various
markers, and observed the complete absence of photoreceptor
markers from the R1/6/7 and R3/4 precursors. Instead, these cells
stained positively for the cone cell marker Cut (Fig. 4E,E⬘) and
for Ttk88 (not shown). Therefore, in the presence of Lz and high
N levels, the R3/4 precursor cells critically require Sev; its
presence specifies them as R7s and its absence directs them to the
non-photoreceptor cone cell fate. This is the exact behavior of the
native R7 precursor cell (Tomlinson and Ready, 1986) and
suggests that the entire R7 specification mechanism can be
recapitulated in the R3/4 cells.
1997; Tang et al., 1997). If the role of Sev activity in the R3/4
transformed cells was indeed to transcriptionally activate phyl, then
supplying phyl transcription independently should replace the need
for Sev. To this end we engineered and transformed a sev.phyl
transgene and checked its efficacy by observing the multiple R7
phenotype in adult ommatidia (Fig. 4G) caused by the
transformation of cone cell precursors into R7 types (Fig. 4H).
When we crossed this into the experimental background (sev0;
sev.N*; sev.lz; sev.phyl), adult sections showed the restitution of R7like cells (Fig. 4J, red arrowheads). In the eye discs, the cells in the
R3/4 positions no longer expressed the cone cell marker Cut, but
showed the characteristic R7 molecular features. Thus, expression
of phyl compensates for the absence of Sev, and suggests that Sev
functions to degrade Ttk by activating phyl transcription.
Specification of the R1/6 type in the Lz-expressing
R3/4 cells
By manipulation of the RTK and N pathways in Lz-expressing R3/4
precursors we had generated the R7 and cone cell fates; the R1/6
fate, however, proved more elusive. Consider sev.lz eyes in which
the R4s are transformed to R7s; we naively expected the R3
precursors to be specified as R1/6 types. This was not the case, as
they expressed the R3/4/1/6 marker Svp but did not express the
R1/6-specific marker Bar. However, R3/4 precursors have a much
higher baseline level of N activity compared with their R1/6
counterparts, as evidenced, for example, by their Sev expression:
Development 140 (21)
high in R3/4, low in R1/6 (Fig. 1D). Furthermore, since sev.lz R3
precursors become R7s, albeit at low frequency, we infer that they
normally experience high N activity, close to that required for R7
specification. We reasoned, therefore, that we should reduce N
activity to allow the emergence of the R1/6 fate.
To reduce N activity we routinely use a sev.Su(H)EnR transgene
that encodes a downregulator of the N pathway expressed under
sev transcriptional control (Tomlinson and Struhl, 2001). The
problem with this transgene is that it downregulates all sevdependent transgenes (including itself – see the Discussion for an
exposition of these effects) including sev.lz, the key construct in
our experiments. We therefore defined a fragment of the rough
(ro) gene that would drive expression in R2,3,4,5 precursors (Fig.
5A), and generated a ro.Gal4 line. When Lz was expressed in this
manner (ro.Gal4; UAS.lz) no adult animals emerged, preventing
an analysis of the mature retina, but in third instar discs many R3/4
precursors showed the R7 transformation phenotype (Fig. 5C).
Interestingly, the R2/5 cells appeared unaffected by their
expression of Lz (as evidenced by normal Ro protein expression;
data not shown). When N signaling was downregulated in the
R3/4 cells [ro.Gal4; UAS.lz; sev.Su(H)EnR], many R3/4
precursors no longer appeared as R7s, but rather expressed both
Svp and Bar – a molecular signature of the R1/6 types (Fig. 5E).
Although in wild type both R3/4 and R1/6 precursors express Svp,
they differ in that R3/4 persistently express it at high levels
whereas in R1/6 its expression decays rapidly and frequently
Fig. 5. The effects of lowering N activity in
sev.lz R3/4 precursors. (A) The GFP staining of a
ro.GFP third instar eye disc is restricted to the
R2/3/4/5 precursors. (A⬘) Additional staining of
Svp and Runt highlights the single R7 and paired
R3/4, R2/5 and R1/6 precursors. (B) Schematic
description of the staining shown in A⬘.
(C,C⬘) A ro.G4; UAS.lz third instar eye disc showing
many cells in the R3/4 positions expressing Runt
and Elav, indicating their differentiation as R7s.
(D) The frequently observed cell fates in ro.G4;
UAS.lz ommatidia. (E,E⬘) When N activity is
downregulated in R3/4 cells in ro.G4; UAS.lz;
sev.Su(H)enR eye discs there is a frequent
transformation of R3 precursors into Barexpressing cells. The circle highlights an
ommatidium in which the cell in the R3 position
expresses Bar and reduced levels of Svp, with its
neighboring R4 differentiating normally and
expressing high levels of Svp (and no Bar). (F) The
cell fates most frequently observed in ro.G4;
UAS.lz; sev.Su(H)enR ommatidia.
Development
4358 RESEARCH ARTICLE
Switching cell fates
RESEARCH ARTICLE 4359
appears at much lower levels than in R3/4. Consistent with these
characteristics, in this experimental background we observed Barexpressing cells in the R3/4 positions that showed the lower
ephemeral Svp level (compare the Svp expression levels in the
Bar-expressing R3/4 cell with the non-Bar-expressing R3/4 cell
in the circled ommatidium in Fig. 5E⬘).
For technical reasons we were unable to evaluate Ro or Spalt
expression to determine whether these cells concomitantly
downregulated R3/4 markers as they expressed the R1/6 signatures.
Accordingly, we focus on the emergence of Bar expression in these
cells as the evidence that they transform into R1/6 types, and infer
that, by the appropriate modulation of the RTK and N signaling
pathways, the R7, R1/6 and cone cell fates could each be reproduced
in Lz-expressing R3/4 cells.
DISCUSSION
In this paper we have explored three inter-related themes bearing
on the nature of the signals that specify the cell types in the
Fig. 6. sev.lz rescue of the lz77a7 mutant. (A) sev.DsRed eye discs
showing the correct sev expression pattern: high in R3/4, R7 and cone
cells but low in R1/6. (A⬘) The same image as A counterstained for Svp
and Runt. (B) In lz77a7eye discs sev.DsRed is initially expressed normally
(high in R3/4 and R7, low in R1/6), but defects become evident as the
strongly expressing cone cells join the cluster. (B⬘) The same image as B
counterstained for Svp. (C) Tangential section through a lz77a7; sev.lz eye,
which is wild type except for the transformation of R3/4 precursors to R7
types (arrowheads). (D) A lz77a7; sev.lz eye disc stained for Svp and Runt
appears normal, except for the expression of Runt in R3/4 cells.
Drosophila ommatidium. We examine the ability of a transcription
factor to predispose the cellular responses to developmental signals,
we validate the accuracy of the signaling code that represents these
developmental signals, and we infer that both the intrinsic and
extrinsic aspects are only required for a brief period of time. Below,
we address each of these themes in sequence.
Lz and the predisposition of second wave cells
Lz had long been assumed to be a key factor that distinguishes how
second wave cells differ from the precluster cells in their response
to developmental signals. In this paper we rigorously tested this
concept and reproduced features typical of the three second wave
cell types in the R3/4 precluster cells by supplying ectopic Lz along
with the appropriate RTK/N cell fate code. We thus infer that the
presence of Lz in R3/4 precluster cells is sufficient to endow them
with the second wave cell fate response repertoire. Below, we
Development
Rescue of lz mutant second wave cells by sev.lz
The sev enhancer element drives expression in a number of cells of
the developing ommatidia, including R3/4, R7 and the cone cells (Fig.
1E). In each of these cells the expression is transitory, occurring for
only a few hours. When the R3/4 precursors are transformed into the
various second wave fates by sev.lz, these transformations are thus
induced by the transient expression of Lz. This suggests that Lz is
only required at the time of cell fate specification and not for the
maintenance of cell fate thereafter. This is surprising because Lz
shows persistent expression in second wave cells, suggesting a
prolonged requirement. To investigate this further we rescued lz gene
function in second wave cells only at the time of their fate
specification. This was achieved by examining the rescuing effects
of sev.lz on lz77a7 second wave cells.
lz77a7 is a mutation caused by the deletion of an eye-specific
enhancer element (Flores et al., 1998; Siddall et al., 2003) that
selectively removes Lz expression from the developing eye, but
leaves it in the antennal disc and throughout the remainder of the
animal, resulting in healthy and fertile animals distinguished only by
severely disrupted eyes (Flores et al., 1998; Siddall et al., 2003).
Although no Lz protein can be detected in lz77a7 eye discs (Flores et
al., 1998) we cannot assume that there is none, and provisionally
regarded it as a strong hypomorph.
We engineered and transformed a sev.DsRed construct to monitor
sev transcription, and confirmed that it showed the correct expression
pattern in a wild-type background: high levels in R3/4/R7/cone cells
and low levels in R1/6 (Fig. 6A). In lz77a7 eye discs, sev.DsRed
expression in the cells in the R1/6/7 positions appeared normal: high
in the R7 precursors and low in the R1/6 cells (Fig. 5B). Thus, the
R1/6/7 precursor cells appear to be recruited to the precluster in a
normal manner even in the absence of Lz. However, even though the
cone cell precursors appear to express high levels of sev, as normal,
they join the cluster in an aberrant manner and from this stage onward
patterning defects were evident.
lz77a7; sev.lz animals showed complete rescue of the second wave
cells, as observed in sectioned adult eyes (Fig. 6C) and in third instar
disc preparations (Fig. 6D). In fact, these eyes appeared completely
wild type, with the exception of the effects engendered by the
ectopic expression of Lz in the R3/4 precursors. Thus, by supplying
lz gene function only at the time when their cell fate decisions are
made, the lz mutant is fully rescued. This provides compelling
evidence that Lz is not required for the later maintenance of the
specified fates.
Development 140 (21)
4360 RESEARCH ARTICLE
discuss a number of issues related to these observations and their
interpretations.
displaying R1/6 molecular features were now detected in the R3/4
precursors.
The N-Dl interaction in sev.lz R3/4 precursors
The insensitivity of R2/5 to ectopic Lz
R7 specification in the absence of sev
Native R7s critically require sev gene function; in its absence, they
differentiate as cone cells. However, some ectopic R7s were able to
differentiate when Lz was provided to the R3/4 precursors, even in
the absence of sev (sev0; sev.lz), suggesting that normal R7
specification was not fully reiterated here. Examination of these eye
discs suggested that some R3/4 precursors differentiated as R7s
whereas others became cone cells. Thus, these cells appear to be on
the cusp of the R7/cone cell fate choice, and we even observed some
cells expressing markers for both cell types. In the cells that became
R7s, we infer the presence of sufficient RTK activity, which was
likely to have been supplied by endogenous DER signaling active
in the precluster cells. Only when N activity was raised in these cells
(sev0; sev.lz; sev.N*) did their full sev dependence for the R7 fate
emerge, when all R3/4 precursors differentiated as cone cells.
Specification of R1/6 types in R3/4 precursors
The sev.N* construct is a very useful activator of the N pathway in
developing eye cells. Since N activity drives sev expression, the
sev.N* transgene feeds back on itself and promotes its own
expression, and by subsequent iterations of this effect the cells are
left with potent N activity. This level is still within the physiological
range, unlike that produced using Gal4/UAS techniques, and is
therefore our choice method for activating the N pathway. The
transgene that we routinely use to knock down N activity
[sev.Su(H)EnR] has the opposite effect; it reduces its own
expression, and mildly compromises N activity. This level of
reduction in N activity is usually sufficient to trigger major effects
without the disadvantage of the severe downregulation that can
accompany the use of Gal4/UAS technology. Since the sev.lz
construct would also be downregulated by sev.Su(H)EnR, we
needed to ectopically express Lz in the precluster using another
enhancer element, and to this end we generated the ro.Gal4 line.
When UAS.lz was expressed under ro control, the R3/4 precursors
frequently differentiated as R7s, and crucially, when N activity was
concomitantly reduced [ro.Gal4; UAS.lz; sev.Su(H)EnR] cells
The cells in the R2/5 positions in ro.G4; UAS.lz developing
ommatidia appear to develop normally; they express Elav and Ro,
but none of the other fate markers. This suggests that R2/5 cells are
insensitive to the presence of Lz, and argues that there is a major
molecular difference between these cells and the R3/4 precursors.
Also noteworthy is the transformation of all lz mutant second wave
cells into R3/4 types characterized by the expression of Svp (a
marker that is not expressed in R2/5 precursors) and Elav. Thus, it
appears that ectopic Lz selectively transforms R3/4 precursors of
the precluster to the second wave fate, and second wave cells
lacking Lz adopt the R3/4 fate. Hence, we suspect that Lz might not
provide the intrinsic information that distinguishes the second wave
cells from precluster cells per se, but rather distinguishes second
wave cells from R3/4 types. We are currently undertaking
experiments to evaluate this view.
A counter-argument emerges from the fate of the majority of cells
in the R3 positions in sev.lz eyes, which do not switch their fate.
Only when we activate or reduce N activity in these cells do we
change their fates, and to be sure that the R2/5 cells are insensitive
to Lz expression we would also need to correspondingly vary N
activity in them. Our experiments to do this using ro.Gal4 produced
severely disrupted preclusters, presumably as a result of interference
with N function at earlier stages of precluster formation. Since these
clusters were largely uninterpretable, the issue of whether R2/5 cells
are insensitive to Lz expression remains unresolved.
Validation of the cell specification code of second
wave cells
For many years, the role of N in photoreceptor specification was
confusing. In some contexts N appeared to oppose photoreceptor
specification and in others N seemed to promote it, and this
confusion prevented substantial progress in defining the fate codes
that specified the different cell types. In recent work we identified
three distinct roles for N in this process, and with that information
inferred the cell fate codes for R1/6, R7 and the cone cells (Fig. 1C).
A major goal of this current work has to been to test this code by its
reiteration in the R3/4 cells of the precluster using Lz expression to
endow them with second wave cell qualities. In these experiments,
each of the cell codes induced the expected cell fates, providing
cogent support for the validity of the code.
A short time window in which Lz and the
developmental signals operate
DER is assumed to be ubiquitously expressed in the eye disc tissue,
and its ligand, Spitz, diffusing from precluster cells, is thought to
reach more distant cells with time. But the N and Sev signals are
regulated in a different manner. Both their ligands are membrane
bound, and their receptor activations only occur in immediate
neighbors. Dl, the ligand for N, is expressed transiently by
differentiating cells, and, accordingly, activates N in neighboring
cells for only short periods of time (a few hours). sev is an N
response gene and, in consequence, Sev is only expressed in cells
for a short period of time. By contrast, Boss, the Sev ligand, is
expressed for a prolonged period by the R8 precursor. Thus, both the
N and Sev signaling systems are only available to the cells for
restricted periods, with this restriction controlled by ligand
expression in the N system and by receptor expression in the Sev
system.
Development
Normal R3/4 precursors undergo an N-Dl interaction that results in
the R4 precursor experiencing much higher levels of N activity than
the R3 precursor (Cooper and Bray, 1999; Fanto and Mlodzik, 1999;
Tomlinson and Struhl, 1999). When Lz was supplied to R3/4
precursors (sev.lz), the cell in the R4 position frequently transformed
into an R7, consistent with the requirement of high N for R7
specification. Less frequently, both R3/4 precursors adopted the R7
fate, and sometimes it was the cell in the R3 position alone that
generated an ectopic R7. These results suggest that in the sev.lz flies
the R3/R4 N-Dl interaction does not occur correctly. When we used
the mδ0.5.lacZ reporter line (Cooper and Bray, 2000) as a reporter
of N activity (which in wild-type larvae is robustly upregulated in
R4 precursors) an erratic pattern was observed, sometimes showing
the wild-type pattern, sometimes showing both R3/4 cells with high
levels of lacZ expression, and sometimes showing R3 alone with
high levels (not shown). Hence, by expressing Lz in the R3/4
precursors we not only endowed them with second wave response
abilities but also prevented them from executing their N-Dl
interactions properly. Indeed, it was only when we artificially
activated N to a high level (sev.lz; sev.N*) that we potently induced
the R7 fate in both R3/4 precursors.
Although Lz is expressed in the second wave cells in a persistent
manner in the eye disc, our experiments suggest that it, like the
extrinsic signals, is required only for a brief developmental window.
Consider the transformation of sev.lz R3/4 cells to the R7 fate. The
sev enhancer is only active in these cells for a few hours and yet a
complete transformation of the cells is achieved. The expression of
specific cell type markers in the eye disc might erroneously indicate
the transformation of a cell when only a transient effect occurs, but
the presence of ectopic R7s in the adult retina argues otherwise and
suggests that the transformations are potent and permanent. This
view is further validated by the rescue of lz mutant second wave
cells by the sev.lz transgene. This rescue is complete and is evident
by the molecular markers expressed in the disc and by the
morphology of the adult cells. Thus, we infer that Lz is only
required during the same time window when the RTK/N signals are
transduced, and we further infer that the combined activities of the
RTK and N pathways, in concert with Lz, function in a short-lived
manner to lock in the fate of the cells. How the presence of
ephemeral extrinsic and intrinsic information is molecularly
‘remembered’ by the cells to allow their appropriate differentiation
over a prolonged developmental period remains an intriguing
question.
Acknowledgements
We thank Jason Rudas for expert technical assistance; Hugo Bellen, Andrea
Brand, Tiffany Cook, Yash Hiromi and John Reinitz for their generous donation
of antibodies; and Jessica Treisman for fly stocks.
Funding
This work was funded by National Institutes of Health grants [R01 EY012536]
to A.T. Deposited in PMC for release after 12 months.
Competing interests statement
The authors declare no competing financial interests.
Author contributions
Y.E.M. and A.T. designed the research, performed experiments, analyzed data
and wrote the paper.
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Development
Switching cell fates